Background: The Repeat Expansion Diseases are caused by intergenerational expansions of a specific tandem repeat. More than 20 such diseases have been identified thus far. The Fragile X-related disorders (FXDs) arise from expansion of a CGG.CCG-repeat in the 5' UTR of the X-linked FMR1 gene. The FXDs are a group of 3 different disorders: Carriers of alleles with 55-200 repeats, so-called premutation (PM) alleles, are at risk for a neurodegenerative disorder, Fragile X-associated tremor-ataxia syndrome and a form of ovarian dysfunction known as FX-associated primary ovarian insufficiency. Furthermore, in females, the premutation allele can undergo expansion on intergenerational transfer that can result in their children having alleles with >200 repeats. This expanded allele is known as a full mutation and individuals who inherit such alleles almost always have Fragile X syndrome (FXS), which is the leading heritable cause of intellectual disability. The mechanism by which is expansion occurs is unknown. It is thought to differ from the generalized microsatellite instability seen in many different cancers in that the instability is confined to a single genetic locus and shows a strong expansion bias. Expanded alleles are also associated with a folate-sensitive fragile site that is coincident with the repeat on the X chromosome. This site, which gives the disorder its name, is one of many fragile sites present on the human genome. These sites are prone to breakage and in some cases are associated with deleterious chromosome deletions and translocations. In the case of the FX fragile site, there is reason to believe that it is responsible for the high frequency loss of the affected chromosome resulting in Turner syndrome (45, X0) in female carriers of a FM allele. Progress report: Using a FX PM model we had previously generated (Entezam et al., 2007), we have shown that the mismatch repair protein MSH2 is absolutely required for all intergenerational and somatic expansions in this model (Lokanga, Zhao and Usdin, 2014). Loss of expansions is associated with an increase in both the number of alleles that have the same number of repeats as the parental allele and alleles that are smaller. The expansion risk in the offspring of parents heterozygous for a null mutation in Msh2, is the same irrespective of the offspring Msh2 genotype. This suggests that most intergenerational expansions occur prezygotically. In the case of maternally transmitted alleles this would indicate that expansion is occurring in the oocyte i.e., at a time when genomic replication is not occurring. This supports our earlier hypothesis that expansion is not related to a problem occurring during normal chromosomal replication but that it arises somehow in non-dividing tissue perhaps related to a problem with DNA repair. Whether MSH2 is acting via a form of mismatch repair to generate expansions or via another DNA repair process in which MSH2 is known to act is currently unknown. We have also shown that males are much more prone to somatic expansion than are females (Lokanga, Zhao, Entezam and Usdin, 2014). This is not due to either a deleterious effect of testosterone in males or a protective effect of estrogen in females. We have shown that females express higher levels of transcripts that encode proteins that protect against oxidative stress. Since we have previously shown that oxidative stress is a risk factor for expansion, the protective effect of these proteins may contribute to the lower level of somatic expansion we observe in females. However, perhaps the most important factor contributing to the difference in expansion frequency is that in females expansion only occurs when the PM allele is on the active X chromosome. The fact that expansion does not occur on the inactive X suggests that expansion requires an open chromatin configuration and/or Fmr1 transcription. This raises the possibility that Transcription Coupled Repair (TCR), a form of nucleotide excision repair that is confined to actively transcribed genes, may contribute to expansion. However, we found that CSB, a protein essential for TCR, only plays an auxiliary role in promoting expansions when other essential factors become rate limiting (Zhao and Usdin, 2014). Thus, it is unlikely that CSB is acting via TCR in this process. Rather our data suggests that it may be promoting expansion via its ability to interact with proteins involved in other DNA repair pathways. We have also extended our earlier work on chromosome fragility to examine the replication of the FMR1 locus in cells from controls, premutation and full mutation carriers. We have shown that while normal alleles have a broad zone of replication initiation that overlaps the 5 end of the FMR1 gene in a variety of cell types, this zone of replication initiation is disrupted in lymphoblastoid cells from individuals with FXS (Yudkin et. al., 2014). This data is consistent with an early observation from our laboratory showing that secondary structures formed by the repeat present a strong barrier to DNA polymerase progression (Woodford and Usdin, 1995; Usdin, 1998). The same abnormal pattern of replication initiation is seen both in untreated cells and when cells are treated with fluorodeoxyuridine, a compound that induces fragile site expression. Thus the expanded repeat tract in FXS cells has difficulty with replication of this region even under normal growth conditions, with FdU-treatment likely causing the appearance of the fragile site by making this region complete replication even more slowly than normal.